Coronavirus Nsp14 modulates the innate immune response

Abstract

Coronavirus (CoV) non-structural protein 14 (nsp14) is encoded by the replicase gene and it is part of the replication-transcription complex. Nsp14 protein is a bifunctional enzyme bearing 3´-5´ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single- and double-stranded RNAs and is part of a proofreading system responsible for the high fidelity of CoV replication. Nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as “non-self” by the host cell. The innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon (IFN) and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but to date the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described. A set of point mutants affecting different motifs within the ExoN domain of nsp14 protein was generated, using transmissible gastroenteritis virus (TGEV) as a model. A specific mutation within zinc finger 1 (ZF-C) led to a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant rTGEVZF-C caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times post-infection. Consequently, the mutant virus triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of IFN-β, TNF, and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced, when compared to the parental virus. Moreover, the mutant virus decreased the antiviral pathway activation produced by the addition of exogenous dsRNA or by the wild-type virus infection, suggesting that the mutant virus actively reduced dsRNA levels in these situations. Overall, our data revealed a novel potential role for CoV nsp14 in modulation of the innate immune response.